We have been investigating the role of Ras proteins and the closely related small molecular weight GTPases in the molecular pathology of two human genetic disorders, neurofibromatosis type 1 (NF1) and tuberous sclerosis (TSC). The product of the neurofibromatosis type 1 (NF1) gene, which is mutated in von Recklinghausen neurofibromatosis, encodes a GAP protein which acts specifically on Ras, converting Ras from the active, GTP- bound form, to the inactive GDP-bound state. Schwann cells are the most abundant cell type in NF1 benign tumors, and thereby contribute to the formation of both benign and malignant tumors in this disease. We have examined the role of the NF1 gene product neurofibromin as a Ras-GTPase activating protein in Schwann cells by isolating Schwann cells from mice with targeted disruption of NF1. The properties of neurofibromin deficient Schwann cells were strikingly similar to those of normal Schwann cells expressing the v-rasH gene, including a marked inhibition of cell growth, morphological changes, and increased levels of Ras*GTP. These studies define a role for neurofibromin as a major regulator of Ras*GTP in normal Schwann cells. Tuberous sclerosis (TSC) is a genetic syndrome characterized by the development of benign tumors in a wide variety of tissues, as well as rare malignancies. The TSC2 gene has been cloned, and encodes an open reading frame with a putative protein product (tuberin) of 1784 amino acids, with a region of limited homology to the catalytic domain of Rap1GAP. We have generated antisera against the N- and C-terminal of tumerin, and these antisera specifically recognize a 180-kDa protein in immunoprecipitation and immunoblotting analyses. Immunoprecipitates of native tuberin from K-562 cells contain an activity that specifically stimulates the intrinsic GTPase activity of Rap1a, and Rap1GAP activity was confirmed in assays with a C-terminal fragment of tuberin, expressed in bacteria or SF9 cells. We conclude that tuberin is a Rap1GAP, and the loss of functional tuberin (which normally would inactivate Rap1) may lead to constitutive activation of Rap1 in tumors of patients with tuberous sclerosis.